Human hematopoietic stem cell

ABSTRACT

Human hematopoietic stem cells are provided by separation of the stem cells from dedicated cells. The stem cells may than be maintained by regeneration in an appropriate growth medium. Means are provided for assaying for the stem cells as to their capability for producing members of each of the hematopoietic lineages.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 07/720,883 filed Jun. 25,1991 which is a continuation-in-part of application Ser. No. 502,616,filed Mar. 30, 1990 now issued as U.S. Pat. No. 5,061,620.

INTRODUCTION

1. Technical Field

The field of this invention is the isolation, regeneration and use ofhematopoietic stem cells.

2. Background

Mammalian blood cells provide for an extraordinarily diverse range ofactivities. The blood cells are divided into several lineages, includinglymphoid, myeloid and erythroid. The lymphoid lineage, comprisingB-cells and T-cells, provides for the production of antibodies,regulation of the cellular immune system, detection of foreign agents inthe blood, detection of cells foreign to the host, and the like. Themyeloid lineage, which includes monocytes, granulocytes, megakaryocytesas well as other cells, monitors for the presence of foreign bodies inthe blood stream, provides protection against neoplastic cells,scavenges foreign materials in the blood stream, produces platelets, andthe like. The erythroid lineage provides the red blood cells, which actas oxygen carriers.

Despite the diversity of the nature, morphology, characteristics andfunction of the blood cells, it is presently believed that there is asingle progenitor, which is capable of self regeneration and by exposureto growth factors becomes dedicated to a specific lineage.

The stem cell population constitutes only a small percentage of thetotal number of leukocytes in bone marrow. In addition, at the presenttime it is not known how many of the markers associated withdifferentiated cells are also present on the stem cell. One marker whichis reported to provide for some enrichment of progenitor activity isClass II HLA (particularly a conserved DR epitope recognized by amonoclonal antibody designated J1-43). However, these markers are foundon numerous lineage committed hematopoietic cells. One marker which hasbeen indicated as present on stem cells, CD34, is also found on asignificant number of lineage committed progenitors. In particular,B-cells (CD19+) and myeloid cells (CD33+) make up 80-90% of the CD34+population. Moreover, a combination of CD3, 8, 10, 15, 19, 20, and 33will mark >90% of all CD34+ cells. Therefore, in view of the smallproportion of the total number of cells in the bone marrow which arestem cells, the uncertainty of the markers associated with the stem cellas distinct from more differentiated cells, and the general inability tobiologically assay for human stem cells, the identification andpurification of stem cells has been elusive. Recently, the mouse stemcell has been obtained in at least highly concentrated, if not apurified form, where fewer than about 30 cells obtained from bone marrowwere able to reconstitute all of the lineages of the hematopoieticsystem of a lethally irradiated mouse. Indeed, one injected cell shouldbe able to reconstitute all of the hematopoietic lineages.

The Thy-1 molecule is a highly conserved protein present in the brainand hematopoietic system of rat, mouse and man. These speciesdifferentially express this antigen and the true function of thismolecule is unknown. However, the Thy-1 molecule has been identified onrat and mouse hematopoietic stem cells. This protein is also present onhuman bone marrow cells and is useful for the selection of hematopoieticstem cells.

There is a strong interest in identifying the human hematopoietic stemcell. Having possession of the stem cell will allow for identificationof growth factors associated with its self regeneration. In addition,there may be as yet undiscovered growth factors associated (1) with theearly steps of dedication of the stem cell to a particular lineage; (2)the prevention of such dedication; and (3) the negative control of stemcell proliferation. The availability of stem cells would be extremelyuseful in bone marrow transplantation, as well as transplantation ofother organs in association with the transplantation of bone marrow.Stem cells are important targets for gene therapy, where the insertedgenes promote the health of the individual into whom the stem cells aretransplanted. In addition, the ability to isolate the stem cell mayserve in the treatment of lymphomas and leukemias, as well as otherneoplastic conditions, e.g., breast cancer. Thus, there have beenworld-wide efforts toward isolating the human hematopoietic stem cell insubstantially pure or pure form.

Relevant Literature

U.S. Pat. No. 4,714,680 describes a composition comprising human stemcells. EPA 89.304651.6 describes the isolation of mouse stem cells. Seealso the references cited therein. Analysis for hematopoieticprogenitors have been reported by Whitlock and Witte, PNAS USA (1982)79:3608; and Whitlock et al., Cell (1987) 48:1009. Thy-1 is a surfacemarker of reconstituting rodent bone marrow stem cells (Berman and BaushExp. Hematol. (1985) 13:1952 and Goldschneider et al., J. Exp. Med.(1978) 148:1351). Muller-Sieburg et al., Cell (1986) 44:653 describeThy-1^(lo) Lin⁻ mouse hematopoietic stem cells and the use of limitdilution.

SUMMARY OF THE INVENTION

Methods resulting in the isolation of substantially homogenouscompositions of human hematopoietic stem cells are provided. The methodsemploy a predetermined separation regimen and bioassays for establishingthe generation of each of the hematopoietic lineages from the isolatedcells. The human stem cells find use: (1) in regenerating thehematopoietic system of a host deficient in stem cells, (2) in a hostthat is diseased and can be treated by removal of bone marrow, isolationof stem cells and treatment of individuals with drugs or irradiationprior to re-engraftment of stem cells, (3) producing varioushematopoietic cells, (4) detecting and evaluating growth factorsrelevant to stem cell self-regeneration; (5) the development ofhematopoietic cell lineages and assaying for factors associated withhematopoietic development; and (6) treatment of genetic diseases throughgene replacement in autologous stem cells.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

A human stem cell composition is provided, substantially free of cellsdedicated to a particular lineage, cells carrying markers associatedwith lineage dedication, wherein the stem cells are able to regenerateand differentiate to populate the various hematopoietic lineages. Thesubstantially homogenous composition may be obtained by selectiveisolation of cells free of markers associated with differentiated cells,while displaying epitopic characteristics associated with the stemcells, and by regeneration of the isolated stem cells in defined culturesystems leading to different hematopoietic cell lineages.

The stem cells are characterized by both the presence of markersassociated with specific epitopic sites identified by antibodies and theabsence of certain markers as identified by the lack of binding ofcertain antibodies. It is not necessary that selection is achieved witha marker specific for stem cells. By using a combination of negativeselection (removal of cells) and positive selection (isolation ofcells), a substantially homogeneous stem cell composition can beachieved.

If desired, a large proportion of differentiated cells may be removed byinitially using a "relatively crude" separation. The source of the cellsmay be the bone marrow, fetal, neonate or adult or other hematopoieticcell source, e.g., fetal liver, peripheral blood, umbilical cord blood,and the like. For example, magnetic bead separations may be usedinitially to remove large numbers of lineage committed cells, namelymajor cell populations of the hematopoietic systems, including suchlineages as T-cells, B-cells, (both pre-B and B-cells), myelomonocyticcells, or minor cell populations, such as megakaryocytes, mast cells,eosinophils and basophils. Desirably, at least about 70%, usually atleast 80% of the total hematopoietic cells will be removed. It is notessential to remove every dedicated cell class, particularly the minorpopulation members at the initial stage. Usually, the platelets anderythrocytes will be removed prior to sorting. Since there will bepositive selection in the protocol, the dedicated cells lacking thepositively selected marker will be left behind. However, it ispreferable that there be negative selection for all of the dedicatedcell lineages, so that in the final positive selection, the number ofdedicated cells present is minimized.

The stem cells are characterized by being for the most part CD34⁺, CD3⁻,CD7⁻, CD8⁻, CD10⁻, CD14⁻, CD15⁻, CD19⁻, CD20⁻, CD33⁻, and Thy-1⁺. Ahighly stem cell concentrated cell composition is CD34⁺, CD10⁻, CD19⁻and CD33⁻, more particularly in addition CD3⁻ and CD8⁻, preferably inaddition Thy-1⁺. The CD3⁻, 8⁻, 10⁻, 19⁻, 20⁻ and 33⁻ will be referred toas Lin⁻. The CD10/19/20 markers are associated with B-cells, CD3/4/8markers are associated with T-cells, CD14/15/33 cell markers areassociated with myeloid cells. The Thy-1 marker is absent on humanT-cells. Also, for human CD34⁺, rhodamine 123 can divide the cells intohigh and low subsets. See Spangrude, (1990) Proc. Natl. Acad. Sci. 87,7433 for a description of the use of rhodamine 123 with mouse stemcells. Preferably the cells are rhodamine low.

In order to obtain the subject stem cells, it is necessary to isolatethe rare pluripotent human stem cell from the other cells in bone marrowor other hematopoietic source. Initially, bone marrow cells may beobtained from a source of bone marrow, e.g., iliac crests, tibiae,femora, spine, or other bone cavities. Other sources of humanhematopoietic stem cells include embryonic yolk sac, fetal liver, fetaland adult spleen, blood, including adult peripheral blood and umbilicalcord blood.

For isolation of bone marrow from fetal bone or other bone source, anappropriate solution may be used to flush the bone, which solution willbe a balanced salt solution, conveniently supplemented with fetal calfserum or other naturally occurring factors, in conjunction with anacceptable buffer at low concentration, generally from about 5-25 mM.Convenient buffers include Hepes, phosphate buffers, lactate buffers,etc. Otherwise bone marrow may be aspirated from the bone in accordancewith conventional ways.

Morphologic evaluation of the 34+Thy+Lin- cells indicates that themultipotent progenitors, "stem cells" are of medium size. Light scatterevaluation shows that "stem cells" have a blast cell profile with lowside scatter. These observations indicate that the "stem cells" have aunique density profile. It has been found that the low density fractionsfrom density fractionated human bone marrow are enriched forCD34+Thy+Lin- cells.

Various techniques may be employed to separate the cells by initiallyremoving cells of dedicated lineage. Monoclonal antibodies areparticularly useful for identifying markers (surface membrane proteins)associated with particular cell lineages and/or stages ofdifferentiation. The antibodies may be attached to a solid support toallow for crude separation. The separation techniques employed shouldmaximize the retention of viability of the fraction to be collected. For"relatively crude" separations, that is, separations where up to 10%,usually not more than about 5%, preferably not more than about 1%, ofthe total cells present having the marker, may remain with the cellpopulation to be retained, various techniques of different efficacy maybe employed. The particular technique employed will depend uponefficiency of separation, cytotoxicity of the methodology, ease andspeed of performance, and necessity for sophisticated equipment and/ortechnical skill.

Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody or used in conjunction with amonoclonal antibody, e.g., complement and cytotoxins, and "panning" withantibody attached to a solid matrix, e.g., plate, or other convenienttechnique. Techniques providing accurate separation include fluorescenceactivated cell sorters, which can have varying degrees ofsophistication, e.g., a plurality of color channels, low angle andobtuse light scattering detecting channels, impedance channels, etc.

One procedure which may be used is in a first stage after incubating thecells from the bone marrow for a short period of time at reducedtemperatures, generally about 4° C., with saturating levels ofantibodies specific for a particular cell type, e.g., CD3 and 8 forT-cell determinants, the cells may then be washed with a fetal calfserum (FCS) cushion. The cells may then be suspended in a buffer mediumas described above and separated by means of the antibodies for theparticular determinants, using various proteins specific for theantibodies or antibody-antigen complex.

Conveniently, the antibodies may be conjugated with markers, such asmagnetic beads, which allow for direct separation, biotin, which can beremoved with avidin or streptavidin bound to a support, fluorochromes,which can be used with a fluorescence activated cell sorter, or thelike, to allow for ease of separation of the particular cell type. Anytechnique may be employed which is not unduly detrimental to theviability of the remaining cells.

Conveniently, after substantial enrichment of the cells lacking themature cell markers, generally by at least about 50%, preferably atleast about 70%, the cells may now be separated by a fluorescenceactivated cell sorter (FACS) or other methodology having highspecificity. Multi-color analyses may be employed with the FACS which isparticularly convenient. The cells may be separated on the basis of thelevel of staining for the particular antigens. In a first separation,starting with at least about 1×10¹⁰, preferably at least about 3×10¹⁰cells, the antibody for CD34 may be labeled with one fluorochrome, whilethe antibodies for the various dedicated lineages may be conjugated to adifferent fluorochrome. Fluorochromes which may find use in amulti-color analysis include phycobiliproteins, e.g., phycoerythrin andallophycocyanins, fluorescein, Texas red, etc. While each of thelineages may be separated in a separate step, desirably the lineages areseparated at the same time as one is positively selecting for CD34 orequivalent marker. Generally, the number of cells obtained will be fewerthan about 1% of the original cells, generally fewer than about 0.5% andmay be as low as 0.2% or less.

The cells may then be further separated by positively selecting forThy⁺, where the cells will generally be fewer than 0.5% of the originalcells, generally in the range of 0.01-0.5%. The cells may be selectedagainst dead cells, by employing dyes associated with dead cells(propidium iodide, LDS). Desirably, the cells are collected in a mediumcomprising 2% fetal calf serum. Other techniques for positive selectionmay be employed, which permit accurate separation, such as affinitycolumns, and the like. The method should permit the removal to aresidual amount of less than about 20%, preferably less than about 5%,of the non-stem cell populations.

The CD34⁺ Lin⁻ and the CD34⁺ Lin⁻ Thy-1⁺ have low side scatter and lowforward scatter profiles by FACS analysis. Cytospin preparations showthe stem cell to have a size between mature lymphoid cells and maturegranulocytes. Cells may be selected based on light-scatter properties aswell as their expression of various cell surface antigens.

While it is believed that the particular order of separation is notcritical to this invention, the order indicated is preferred.Preferably, cells are initially separated by a coarse separation,followed by a fine separation, with positive selection of a markerassociated with stem cells and negative selection for markers associatedwith lineage committed cells. This separation is followed by selectionfor a cellular composition having multi-lineage potential and enhancedself-regeneration capability.

Compositions having greater than 90%, usually greater than about 95% ofhuman stem cells may be achieved in this manner, where the desired stemcells are identified by being CD34⁺, Lin⁻ and Thy-1⁺ and being able toprovide for cell regeneration and development of members of all of thevarious hematopoietic lineages. Ultimately, a single cell could beobtained from a stem cell composition and be used for long termreconstitution of an immunodeficient human, if one could be assured thatthe cell was located in the proper environment in vivo.

The subject compositions are found to provide for production of myeloidcells and lymphoid cells in appropriate cultures, cultures providinghydrocortisone for production of myeloid cells (associated withDexter-type cultures) and B lymphocytes in cultures lackinghydrocortisone, (associated with Whitlock-Witte type cultures). In eachof the cultures, mouse or human stromal cells are provided, which maycome from various strains, AC3 or AC6, stromal cells derived from mouseor human fetal bone marrow by selection for the ability to maintainhuman stem cells, and the like. The medium employed for the culturing ofthe cells is conveniently a defined enriched medium, such as IMDM(Iscove's Modified Dulbecco's Medium), a 50:50 mixture of IMDM and RPMI,and will generally be composed of salts, amino acids, vitamins, 5×10⁻⁵ M2-ME, streptomycin/penicillin and 10% fetal calf serum, and may bechanged from time to time, generally at least about once to twice perweek. Particularly, by transferring cells from one culture withhydrocortisone, to the other culture without hydrocortisone, anddemonstrating the production of members of the different lineages in thedifferent cultures, the presence of the stem cell and its maintenance issupported. In this manner, one may identify the production of bothmyeloid cells and B-cells.

To demonstrate differentiation to T-cells, one may isolate fetal thymusand culture the thymus for from 4-7 days at about 25° C., so as tosubstantially deplete the lymphoid population of the fetal thymus. Thecells to be tested are then microinjected into the thymus tissue, wherethe HLA of the population which is injected is mismatched with the HLAof the thymus cells. The thymus tissue may than be transplanted into ascid/scid mouse as described in EPA 0 322 240, particularlytransplanting in the kidney capsule.

For red blood cells, one may use conventional techniques to identifyBFU-E units, for example methylcellulose culture (Metcalf (1977) In:Recent Results in Cancer Research 61. Springer-Verlag, Berlin, pp 1-227)demonstrating that the cells are capable of developing the erythroidlineage.

In identifying myeloid and B-cell capability, conveniently, thepopulation to be tested is introduced first into a hydrocortisonecontaining culture and allowed to grow for six weeks in such culture.The medium employed will comprise a 50:50 mixture of RPMI 1640 and IMDMcontaining 10% FCS, 10% horse serum, streptomycin/penicillin, glutamineand 5×10⁻⁷ M hydrocortisone. During the six week period, it would beanticipated that in the absence of progenitor cells, all of the maturecells would die. If at the end of six weeks, myeloid cells are stillobserved, one may conclude that there is a progenitor cell which isproviding for the continuous differentiation to myeloid cells. At thistime, one may then change the medium, so that the medium now lackshydrocortisone, to encourage the growth of B-cells. By waiting 3-4 weeksand demonstrating the presence of B-cells by FACS analysis, one mayconclude that the progenitor cells which previously were capable ofproducing myeloid cells are also capable of producing B-cells. Humanhematopoietic cells grown in the presence of hydrocortisone can bemaintained for at least four months. Similarly, human hematopoieticcells grown in the absence of hydrocortisone contain B lymphocytes(CD19⁺), as well as myelomonocytic cells for at least four months. Fromthese cultures, one may sort for CD34+ Lin⁻, CD34+ Thy+, Thy+Lin⁻, orCD34+ Thy+ Lin⁻, which should provide a composition substantiallyconcentrated in the progenitor hematopoietic stem cell. The CD34+Lin⁻,CD34+Thy+, Thy+Lin⁻, or CD34+Thy+Lin⁻ cells obtained from these culturescan give rise to B-cells, T-cells and myelomonocytic cells in the assaysdescribed above.

A pluripotent human stem cell may be defined as follows: (1) gives riseto progeny in all defined hematolymphoid lineages; and (2) limitingnumbers of cells are capable of fully reconstituting a seriouslyimmunocompromised human host in all blood cell types and theirprogenitors, including the pluripotent hematopoietic stem cell by cellrenewal. In the subject compositions, fewer than a total of about 10⁷cells, usually fewer than 10⁶ cells, may be used to reconstitute animmunocompromised human host, as compared to the number of stem cellsincluded in whole bone marrow transplants (˜10⁷). The number of cells isrequired to insure that appropriate seeding at an appropriate siteoccurs, where the stem cell may self-renew. The number of cells requiredwhich become seeded at a proper site for self-renewal may be fewer than50 cells, and as few as about a total of 20 cells or fewer, are able tofulfill the conditions indicated above. Thus, based on the standards setfor the earliest progenitor pluripotent stem cell, the subjectcompositions are capable of fulfilling these requirements. Furthermore,the subject cells based on analysis of bone marrow cells appear to be ina range of from about 0.01-0.1% of bone marrow cells, particularly0.01-0.05%.

Once stem cells have been isolated, they may be propagated by growing inconditioned medium from stromal cells, such as stromal cells that can beobtained from bone marrow, fetal thymus or fetal liver, and are shown toprovide for the secretion of growth factors associated with stem cellmaintenance, coculturing with such stromal cells, or in mediumcomprising maintenance factors supporting the proliferation of stemcells, where the stromal cells may be allogeneic or xenogeneic. Beforeusing in the coculture, the mixed stromal cell preparations may be freedof hematopoietic cells employing appropriate monoclonal antibodies forremoval of the undesired cells, e.g, with antibody-toxin conjugates,antibody and complement, etc. Alternatively, cloned stromal cell linesmay be used where the stromal lines may be allogeneic or xenogeneic.

The subject cell compositions may find use in a variety of ways. Sincethe cells are naive, they can be used to reconstitute fully anirradiated host and/or a host subject to chemotherapy; or as a source ofcells for specific lineages, by providing for their maturation,proliferation and differentiation into one or more selected lineages byemploying a variety of factors, such as erythropoietin, colonystimulating factors, e.g., GM-CSF, G-CSF, or M-CSF, interleukins, e.g.,IL-1, -2, -3, -4, -5, -6, -7, -8, etc., Leukemia Inhibitory Factory(LIF), Steel Factor (Stl), or the like, or stromal cells associated withthe stem cells becoming committed to a particular lineage, or with theirproliferation, maturation and differentiation. The stem cells may alsobe used in the isolation and evaluation of factors associated with thedifferentiation and maturation of hematopoietic cells. Thus, the stemcells may be used in assays to determine the activity of media, such asconditioned media, evaluate fluids for cell growth activity, involvementwith dedication of particular lineages, or the like.

The stem cells may be used for the treatment of genetic diseases.Genetic diseases associated with hematopoietic cells may be treated bygenetic modification of autologous or allogeneic stem cells to correctthe genetic defect. For example, diseases such as B-thalassemia, sicklecell anemia, adenosine deaminase deficiency, recombinase deficiency,recombinase regulatory gene deficiency, etc. may be corrected byintroduction of a wild-type gene into the stem cells, either byhomologous or random recombination. With allogeneic stem cells, normalcells lacking the genetic defect can be used as a therapy. Otherindications of gene therapy are introduction of drug resistance genes toenable normal stem cells to have an advantage and be subject toselective pressure, e.g. the multiple drug resistance gene (MDR).Diseases other than those associated with hematopoietic cells may alsobe treated, where the disease is related to the lack of a particularsecreted product such as a hormone, enzyme, interferon, factor, or thelike. By employing an appropriate regulatory initiation region,inducible production of the deficient protein may be achieved, so thatproduction of the protein will parallel natural production, even thoughproduction will be in a different cell type from the cell type thatnormally produces such protein. It is also possible to insert aribozyme, antisense or other message to inhibit particular gene productsor susceptibility to diseases, particularly hematolymphotropic diseases.

Alternatively, one may wish to remove a particular variable region of aT-cell receptor from the T-cell repertoire. By employing homologousrecombination, or antisense or ribozyme sequence which preventsexpression, the expression of the particular T-cell receptor may beinhibited. For hematotropic pathogens, such as HIV, HTLV-I and II, etc.the stem cells could be genetically modified to introduce an antisensesequence or ribozyme which would prevent the proliferation of thepathogen in the stem cell or cells differentiated from the stem cells.

Methods for recombination in mammalian cells may be found in MolecularCloning, A Laboratory Manual (1989) Sambrook, Fritsch and Maniatis, ColdSpring Harbor, N.Y.

The cells may be frozen at liquid nitrogen temperatures and stored forlong periods of time, being thawed and capable of being reused. Thecells will usually be stored in 10% DMSO, 70% autologous plasma(irradiated with 2500 rad), 20% Tc199 (Tissue culture medium). Cells arefrozen in a programmable cell freezer to -180° C. in liquid nitrogen.Once thawed, the cells may be expanded by use of growth factors orstromal cells associated with stem cell proliferation anddifferentiation.

The hematopoietic stem cells, either autologous or allogeneic, may beused for treatment of various diseases where toxic therapies may beinvolved. For example, in the treatment of neoplasia, bone marrow may beremoved from the patient (autologous) or from a "matched" donor("allogeneic") and the stem cells isolated and optimally frozen. Thepatient's bone marrow may be partially or wholly ablated usingirradiation and/or chemotherapy. Once the treatment is completed, thestem cells may be thawed, if appropriate, administered to the patient byany convenient means, e.g., intravascularly, in a physiologicallyacceptable medium. The patient may then be monitored for signs ofengraftment.

The stem cells may be grown in culture, whereby the stem cells may beexpanded. In this way, one can repetitively administer stem cells duringa course of a toxic therapy.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

Materials and Methods

Antibodies. The antibodies to the various markers were obtained asfollows: CD3, 8, 10, 14, 15, 19, 20, 33, 34 (Becton-Dickinson) CD33(Coulter Immunology), (Dalchau and Fabre, J. Exp. Med. (1979) 149:576).The CD34 antibody (IgG3) Tuk3 obtained from A. Ziegler (in LeukocyteTyping IV; White cell differentiation antigens 1989, 817). The CD3, -8,-10, -14, -15, -19, -20, -33 were purchased as FITC conjugates. Theantibody from Ziegler was detected using the appropriate anti-IgG3conjugated to fluorescein (FL), or phycoerythrin (PE) or Texas red (TR)(Caltag). The Thy-1 antibody was a fluorescein, phycoerythrin or biotinconjugate, where the biotin conjugate was detected with TR, FL orPE-avidin (Caltag).

Fluorescence Activated Cell Sorter (FACS) Analysis and Sorting.

A Becton-Dickinson FACS modified as described (Parks and Herzenberg,Meth. Enzymol. (1984) 108:197) was employed. The dual laser instrumentallows four fluorescent parameters and two light scatter parameters tobe recorded for each analyzed cell. Residual erythrocytes and dead cellsand debris were excluded from analysis by light scattering gating and PI(propidium iodide) staining or by scattering alone in 4-color analyses.Compensation for spatial overlaps of fluorescein and phycoerythrin, andfluorescein and propidium iodide was adjusted electronically asdescribed (Parks and Herzenberg, (1984) supra). Four color stains wereperformed using several combinations of the same reagents conjugated todifferent fluorochromes to assure that the results were consistentregardless of the various spatial overlaps of the fluorochromes. Inaddition, the results of 4-color analyses were calibrated by comparisonwith data from 2- and 3-color analyses.

For cell sorting, the stained samples were maintained at 4° C.throughout the sorting procedure. Sorted drops were collected in RPMI1640 containing 10% fetal calf serum (Hazelton Biologics Inc., Lenexa,Kans.). Two color sorts employed phycoerythrin to label CD34 andfluorescein to label LIN cells, with propidium iodide (PI) to label deadcells, with both signals being detected and excluded in a single FACSchannel. Three color sorts employed Texas red to label CD34,phycoerythrin to label Lin cells and fluorescein to label Thy-1 cells.Following isolation of a cell population by FACS, the sample was diluted1:1 in HBSS, centrifuged for 10 minutes at a RCF of 200 and resuspendedin 50 or 100 μl of HBSS for hemocytometer counting.

The culture assays were performed as follows:

Various murine stromal cell lines were employed, three of which aredescribed in Whitlock et al., Cell (1987) 48:1009-1021. Confluentstromal cell layers were maintained for up to 3-4 weeks without passageby changing of the tissue culture medium every 5-7 days. To passage, thestromal cell layers were washed 3 times with serum-free medium, thenoverlaid with 2.5 ml (T-25 flask) of 0.5 mg/ml collagenase-dispase(Boehringer-Mannheim, Indianapolis, Ind.) in serum-free medium. Thecultures were allowed to incubate 15-30 minutes at 37° C.; then thecells in the enzyme-containing medium were collected and RPMI-1640medium with serum added. The stromal cells were suspended by pipettingwith a Pasteur pipet, then cultured directly at 1-5th to 1-50th theoriginal cell concentration. In general, confluent stromal layerssubcultured at 1:10 reached confluency again after 5-7 days. Subcloneswere obtained by limiting dilution culture from 30 to 0.3 cells perwell. Human stromal cell lines were treated similarly.

Cell suspensions of human fetal bone marrow were prepared from longbones of fetuses from 10-18 week gestation. The bones are splitlengthwise and the medullary cavity is scraped with a scalpel blade. Thebones are then placed in a 1 mg/ml solution of collagenase/dispase inRPMI-1640. The bones are incubated for 30 minutes at 37° C., after whichtime the medullary cavity is flushed with media (RPMI-1640 withPen/Strep, 2-ME and 5% FCS) to remove hematopoietic cells.Alternatively, bone marrow may be flushed from the marrow cavity withoutcollagenase/dispase treatment.

Cell suspensions are prepared from livers of 16-20 week gestationfetuses. The liver is minced and then pipetted to release cells. Thecell suspension is then placed on a Ficoll gradient to removehepatocytes, red blood cells and debris. The hematopoietic cells arethen harvested.

Adult bone marrow is obtained from marrow aspirates, which are treatedto remove red blood cells before use.

Bulk cultures are obtained by placing the human cells on the previouslyestablished confluent layer of mouse or human stromal cell lines. From3×10⁴ to 2×10⁵ cells per ml are placed on the stromal cells in eitherT-25 flasks or 6 well plates, by addition of 3 ml to each well of a 6well plate or 5 ml to T-25 flask. A 50:50 mixture of RPMI-1640 and IMDMcontaining 50 μg/ml penicillin/50 μg/ml streptomycin, 1 mM sodiumpyruvate, 2 mM glutamine, 5×10⁻⁵ 2-mercaptoethanol and 10% fetal calfserum is employed. For Dexter-type conditions, IMDM containing 50 μg/mlpenicillin/50 μg/ml streptomycin, 1 mM sodium pyruvate, 2 mM glutamine,10% fetal calf serum, 20% horse serum and 10⁻⁶ M hydrocortisone sodiumsuccinate is employed. Bone marrow cells grown in the Dexter-type mediumgive rise only to myeloid differentiation. Cultures were establishedwith whole cell populations or cells fractionated by their expression ofcell surface antigens (CD34, HLA-DR, Thy-1, Lineage markers).

Limiting dilution cultures were prepared using 96 well plates containingthe mouse stromal cells as confluent layers. The human cells weretitered into the plates at progressively lower concentrations with atleast 24 wells plated at each cell concentration. The plates were thenexamined to determine the percentage of positive wells at each cellnumber. The data is then plotted graphically.

AC3 and AC6 Cocultures

Cocultures established with the mouse bone marrow stromal cell lines,AC3 or AC6, have served successfully as feeder layers for human culturesand have inhibited fibroblast overgrowth at low cell densities.

(1) Cell suspensions from more than one hundred human fetal bone marrow,fetal liver, or adult bone marrow samples have been cocultured for up to20 weeks with continuous production of hematopoietic cells during thistime indicating that early human progenitors or stem cells have beenestablished in these cultures.

(2) Cultures show small to medium sized human bone marrow cells attachedto the mouse stromal cells and proliferation occurs over the first oneto three weeks of culture; thereafter they remain fairly stable.

(3) Cells form loose aggregates consisting of non-adherent and adherentcells overlying stromal cells which in turn overlie small tointermediate sized cells (pseudo-emperipolesis). Overall, the appearanceof the cultures is similar to mouse long-term cultures.

(4) Cytospin and Fluorescence Activated Cell Sorter (FACS) analyses showmaintenance of human hematolymphoid cells. In the absence ofhydrocortisone (Whitlock-Witte like conditions), cultures are a mixtureof myeloid, monocytoid, and lymphoid lineages by morphology. Themajority of cells are myeloid and vary from myeloblasts to maturepolymorphonuclear cells. From 15-40% of the cells are mononuclear andmany of these cells have a lymphoid morphology.

(5) Approximately 20-40% of the cells stain with the CD15 antibody and10-50% of the cells stain with the B lineage markers, CD10, CD19, orCD20, indicating a significant number of B-cells. Cells selected fromfetal bone marrow for CD19 expression survive for less than three weeksin culture. The presence of CD10⁺ and CD19⁺ cells after >4 weeks inculture indicates that early B-cells are arising from committedprogenitors. In addition, from 1 to 10% of the cells stain forcytoplasmic μ heavy chain which confirms the presence of pre-B-cells.Significant numbers of cells express CD20 although fewer than 1% expresssIg, indicating that few of the early B-cells mature under the indicatedculture conditions. Furthermore, cultures initiated after depletion ofB-cells (CD10, CD19) by cell sorting show B-cell development within oneweek of culture initiation.

(6) Cultures initiated in the presence of hydrocortisone (Dexter-likeconditions) have no detectable T- or B-cells and have a large percentageof granulocytes and myeloid cells as evidenced by FACS analysis andcytospins. The presence of mitotic figures and the long term maintenanceof these cultures indicates the presence of some active progenitor cell.When these hydrocortisone containing cultures are switched to mediawithout hydrocortisone, 2 to 6% CD19 cells can be found within twoweeks. This data further substantiates the presence of an activeprogenitor cell in this coculture system.

Using the above defined co-culture system, a limit dilution assay hasbeen developed which can be used to determine the frequency of colonyforming cells in the various cell subpopulations. The frequency isdetermined by the cell number at which 37% of the wells show nocolonies. One representative experiment showed that 1/2000 whole bonemarrow cells responded while 1/200 and 1/30,000 cells responded in theCD34+ and CD34- subsets respectively.

In addition, a single cell assay has been developed in which single FACSsorted progenitor cells are placed into individual wells of a 96 wellplate containing a mouse or human bone marrow stromal cell feeder layer.Results shown on Table 7 indicate that 1 in 40 CD34+LIN⁻ (Lin=CD3, 10,19, 20, 15, 33) or 1 in 80 CD34+Lin+ cells respond by colony formation(0.5-1% of bone marrow). Analysis of the colonies show that 40% and 25%of the colonies are multipotent as determined by FACS andmethylcellulose analysis (B lymphoid, myeloid, erythroid) in theCD34+Lin- and CD34+Lin+ populations respectively. Further, CD34+Thy+cells (0.1-0.5% of bone marrow) show colony growth in 1 in 20 wells. Themajority of the colonies are multipotent (>70%). The CD34+Thy+ cells arethe most efficient population in terms of growth after transfer to newstromal layers. In contrast, the CD34+Thy- cells, which represent >90%of the CD34+ cells, respond poorly in the co-culture assay; 1 in 400cells form colonies, none of which are multipotent. The Thy+ populationcan also be subdivided according to expression of mature lineagemarkers. The Thy+Lin- cell subset (0.1-0.5% of bone marrow) respondswell in the single cell assay (1/20) while the Thy+Lin+ subsets respondspoorly (0/800). FACS and methylcellulose assays show that >70% of thecolonies derived from Thy+Lin- cells are multipotent. The above dataindicates that "stem cells" are present in the subset of cells whichexpress CD34 and Thy-1 but lack expression of lineage markers. Cellswith this phenotype represent fewer than 1 in 1000 whole bone marrowcells.

One can determine the frequency of cells in the starting populationwhich grow under the above defined conditions. The frequency isdetermined by the cell number at which 37% of the wells show no growth.In one study, 1/1500 of the unsorted cells respond while 1/40 of theCD34⁺ fraction and 1/4000 of the CD34- fraction respond.

The following tables indicate the results for the in vitro cultureassays.

                                      TABLE 1                                     __________________________________________________________________________    ANTIGEN STAINING PROFILE OF NON-ADHERENT HUMAN                                CELLS GROWN IN THE CO-CULTURE SYSTEM WITHOUT HYDROCORTISONE                       Time in         CD34                                                      Sample                                                                            Culture                                                                           CD10                                                                              CD19                                                                              CD20                                                                              (progenitor                                                                         CD15 Methylcellulose                                #   (Weeks)                                                                           (B-cell)                                                                          (B-cell)                                                                          (B-cell)                                                                          cell) (myeloid)                                                                          (col/10.sup.5)                                 __________________________________________________________________________    S133                                                                              0                          200                                                7   24  14  ND  38    ND                                                      9   ND  7   12  31    ND   92                                                 12  45  29  33  46    45   115                                                14  21  8   13  18    22   120                                            S143                                                                              0                          156                                                3   31  18  15  36    40   84                                                 5   39  21  34  65    42   96                                                 8   19  15  45  51    ND   44                                                 10  24  26  19  41    31   140                                            __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    BONE MARROW UNDER DEXTER CONDITIONS                                           Time in                                                                            culture                                                                            CD15 CD19  CD33 CD34                                                Sample#                                                                            (weeks)                                                                            Condition                                                                          (mono/gran)                                                                         (B-cell)                                                                           myeloid                                                                            (progenitor cell)                              __________________________________________________________________________    K146 5    +HC  93.5   0.23                                                                              40.9 20.6                                           K146 9    +HC  91.5  ND   90.5 68.3                                           K146 7/2* +HC/-HC                                                                            84.6  6.0  ND   58.4                                           K146 10   +HC  95.0  0.3  26.0 23.0                                           __________________________________________________________________________     *Switch cultures  initiated in hydrocortisone, switched to media without      hydrocortisone.                                                          

FACS separation of Fetal Bone Marrow (FBM) was performed dividingfractions into CD34⁺, 10⁺, 19⁺ and CD34⁺ 10⁻, 19⁻. The fractions arethen grown continuously in the absence of hydrocortisone for eight weeksand screened for the presence of myeloid cells and B-cells.

                  TABLE 3                                                         ______________________________________                                        KINETICS OF B-CELL GENERATION IN VITRO AFTER                                  FACS DEPLETION OF B-CELLS (CD10+, CD19+)                                      FROM FETAL BONE MARROW                                                                Cell subset                                                           Days in vitro                                                                         (K336)    CD10   CD15 CD19 k/1   CD33 CD34                            ______________________________________                                        d8      WBM       72%    43%  65%  5%/4% 25%   4%                                     34+10+19+ 95     14   95   4/1    5    4                                      34+10-19- 40     61   32   0.1/0.1                                                                             65    6                              d15     WBM       31     34   42   6/8   37    3                                      34+10+19+ 90      6   88   11/20 12    1                                      34+10-19- 16     38   18   1/2   38    2                              d22     WBM       28     37   21   1/3   70    2                                      34+10+19+ 20     ND*  20    4/12 ND*  ND*                                     34+10-19- 31     43   23   1/2   73    4                              d29     WBM       49     39   32   1/2   54   21                                      34+10+19+ ND*    ND+  15   3/9   77   ND*                                     34+10-19- 73     21   66   1/2   31   13                              d56     WBM       ND     19    7   0/0   81    3                                      34+10+19+ ND      2    0   0/0   50    4                                      34+10-19- ND     15   23   1/1   69    2                              ______________________________________                                         Numbers indicate the percentage of cells stained by the indicated MAb as      determined by FACS.                                                           *Indicates insufficient cells for analysis                               

Alternatively, fetal WBM is separated by FACS into CD34⁺, 33⁻, 10⁻, 19⁻and the cells are grown in the absence of hydrocortisone. By employinglimit dilution about 10-100 cells are found to be able to be maintainedin the coculture for greater than six weeks and be differentiated intomature myeloid and B-cells.

                                      TABLE 4                                     __________________________________________________________________________    ANALYSIS OF B-LYMPHOID AND MYELOID CELLS                                      IN VITRO CULTURES                                                             Cells Time                                                                              % CD10 % CD19                                                                              % k/1 % CD15                                                                             % CD34                                      (K283)                                                                              (days)                                                                            (Early B-cell)                                                                       (Pan-B-cell)                                                                        (Mature B)                                                                          (Myeloid)                                                                          (Progenitor)                                                                        GM Colonies 10.sup.5                                                                  BFU-e/10.sup.5                __________________________________________________________________________    WBM   0   35     33    7/3   45   8     59      89                            34+10+33+                                                                           0   70     60    2/1   35   98    1199    755                           34+10-33-                                                                           0   1      3     0.1/0.1                                                                             1    95    72      102                           WBM   21  25     14    2.2/1.9                                                                             20   0.13  39      39                            34+10+33+                                                                           21  12     1.7   0.2/0.2                                                                             39   1.9   1976    1576                          34+10-33-                                                                           21  52     49    8.2/4.9                                                                             31   0.4   1841    1685                          __________________________________________________________________________

The results of the CD34, 33, 10 separation are shown in Table 4. Thesorted cell populations as well as the unsorted cells were analyzed atthe time of separation (t=0) as well as twenty-one days (t=21) later.Analysis of the FACS staining profile at t=0 shows that myeloid andB-cells were effectively removed from the CD34⁺ CD10⁻ CD33⁻ cells withless than 5% contaminating B and myeloid cells. In contrast, by day 21about 50% of the CD34⁺ CD10⁻ CD33⁻ cells were B-cells. In addition,there was a ten-fold increase in cell numbers between t=0 and t=21.Therefore, there was an overall 100-fold increase in B-cells over the 21day period. Further, about one-third of the B-cells express sIg with a2/1 ratio of kappa and lambda light chains. The results indicate thatthe B-cells are polyclonal and do not represent an Epstein-Barr viraltransformation. In comparison, the CD34⁺ CD10⁺ CD33⁺ cells show adramatic decrease in B-cells and total cell numbers over the 21 dayperiod. The results show that the CD34⁺ cells which express CD10 and/orCD19 are not long lived progenitors.

Myeloid cell differentiation was analyzed by FACS and methylcelluloseassay. FACS analysis shows a 10 fold increase in mature myeloid cells inthe CD34⁺ CD10⁻ CD33⁻ cell subset. Analysis of the methylcellulose datashowed that 90-95% of the CFU-GM and BFU-e activity is contained in theCD34⁺ CD10⁺ CD33⁺ cell subset at time zero. However, at day 21, theCD34⁺ CD10⁺ CD33⁺ and the CD34⁺ CD10⁻ CD33⁻ cell populations have nearlyequivalent CFU-GM and BFU-e cell levels. Therefore, the CD34⁺ CD10⁻CD33⁻ cells have the capacity to give rise to B-cells (CD19⁺,cytoplasmic μ⁺, sIg⁺), myeloid cells (CD15⁺, -33⁺, CFU-GM) and erythroidcells (BFU-e) over 21 days in culture. Similar results are obtained whencells are separated on the basis of CD34,CD10,CD19,CD33 orCD34,CD3,CD7,CD8,CD10, CD14,CD15,CD19,CD20,CD33.

When CD34⁺ cells are divided into Thy-1⁺ and Thy-1⁻ fractions andassayed in the methylcellulose assay, these two fractions give similarreadouts as seen in Table 5. When assayed by limiting dilution and inthe cocultures, the CD34⁺,Thy-1⁺ fraction is enriched for progenitoractivity as evidenced by 1) the percentage of B-cells generated in thecoculture; 2) the frequency of responding cells in the limit dilutionassay and 3) the frequency of responding cells in the single cell assay(Tables 5 and 6). In order to obtain 75% positive wells in the limitdilution assay, the Thy-1⁺ fraction required approximately 1/30-1/50cells whereas the Thy-1⁻ fraction requires 1/170-1/500 cells. Thisindicates that the Thy-1⁺ fraction is further concentrated for theprogenitor cell. Moreover, when assayed in the in vivo T-cell assay, theThy-1⁺ fraction gives rise to donor derived T-cells, whereas the Thy-1⁻fraction does not.

                  TABLE 5                                                         ______________________________________                                        FREQUENCY ANALYSIS OF CD34+ THY1 POPULATIONS                                  IN METHYCELLULOSE AND LIMIT DILUTION ASSAYS                                          Methycellulose                                                                          Limit Dilution Frequency                                     Sample   Freq. of GM col                                                                           21 days   28 days                                                                             35 days                                  ______________________________________                                        K275 WBM   1/10,000  NA         1/3000                                                                             NA                                       CD34+Thy+                                                                              1/284       "         1/35  "                                        CD34+Thy-                                                                              1/312       "         1/170 "                                        K306 WBM  1/1430     NA        NA    NA                                       CD34+Thy+                                                                              1/100       1/120     1/50  NA                                       CD34+Thy-                                                                              1/340       <1/1000   1/500 <1/800                                   ______________________________________                                    

                                      TABLE 6                                     __________________________________________________________________________    ANALYSIS OF CD34/THY-1 SUBPOPULATIONS IN                                      LONG TERM BULK CULTURES (10.sup.5 CELLS/WELL)                                             % CD10                                                                             % CD19         CD34                                                 Time in                                                                            (early                                                                             (early                                                                             % CD15                                                                             % CD33                                                                             (progenitor                                   Cell Pop.                                                                            Culture                                                                            B-cell)                                                                            B-cell)                                                                            (myeloid)                                                                          (myeloid)                                                                          cell)                                         __________________________________________________________________________    K299 WBM                                                                             8    0.1  0.1  5.4  60.8 1.0                                           CD04+Thy1+                                                                           8    31.0 37.0 42.7 20.2 0.3                                           CD34+Thy1-                                                                           8    0.5  0.4  9.0  28.1 0.4                                           K332 WBM                                                                             7    0.3  0.4  1.9  80.4 13.0                                          CD34+Thy1+                                                                           7    49.0 43.4 20.0 57.7 3.5                                           CD84+Thy1-                                                                           7    7.4  8.2  3.1  76.4 2.6                                           __________________________________________________________________________

The thymus assay for T-cell generation was performed as follows. Fetalthymus fragments (individual thymic lobes) are obtained of about 1 mm³in size. The fragments are cultured in a thymus organ culture system at25° C. for 3-7 days to stimulate the in vitro receptivity of the thymusfor precursor cells.

The cell composition comprising about 10⁰ -10⁴ cells in a FCS containingbalanced salt solution is injected at a volume of 1 μl using a glassmicropipet linked to an oil-filled micrometric screw-operated syringe.Twenty-four hours after injection, the in vitro colonized thymusfragments are implanted under the kidney capsule of SCID mice. See EPA 0322 240. The injected cells are HLA mismatched with the thymus. Atintervals, recipient animals are sacrificed and the grafts harvested.Cell suspensions are analyzed in a two-color immuno-fluorescence assayfor the presence of donor derived T lymphocytes (CD3⁺, 8⁺).

                  TABLE 7                                                         ______________________________________                                        SINGLE CELL PER WELL EXPERIMENTS                                                           FREQUENCY OF  FREQUENCY OF                                       BONE MARROW  GROWTH        MULTIPOTENT                                        SUBPOPULATION                                                                              POSITIVE WELLS                                                                              COLONIES                                           ______________________________________                                        CD34+Lin+    1/80*         25%**                                              CD34+Lin-    1/40          40                                                 CD34+Thy+    1/20          75                                                 CD34+Thy-    1/400          0                                                 Thy+Lin+     0/854          0                                                 Thy+Lin-     1/21          76                                                 ______________________________________                                         Single cells with the described phenotype were deposited in individual        wells of a 96 well plate which contained a bone marrow stromal cell feede     layer.                                                                        *Numbers represent the frequency of cells which gave rise to colonies of      >100 cells. The numbers represent the average of at least three separate      experiments constituting at least 10 96 well plates.                          **Numbers represent the percentage of colonies arising from single cells      which contained cells of the Blymphoid (CD10+, CD19+), Myeloid (CD15+,        CD33+, GMCFU) and erythroid (BFUe) lineages simultaneously.              

                  TABLE 8                                                         ______________________________________                                        T-cell repopulation of the human thymus                                       following in vitro colonization by microinjection                             of leukocyte/precursor cells and regrafting into SCID mice                    Precursor Cell                                                                             Microinjected                                                                            Repopulation                                          Phenotype    Cell Number                                                                              (+/Total)                                             ______________________________________                                        (FBM) CD34.sup.+                                                                           10.sup.4    9/11                                                 (FBM) CD34.sup.+                                                                           10.sup.2   2/4                                                   (FBM) CD34.sup.+, 7.sup.-                                                                  10.sup.4   6 wks: 3/5; 11 wks: 2/3                               (FBM) CD34.sup.+, 7.sup.+                                                                  10.sup.4   6 wks: 3/4; 11 wks: 0/3                               (FBM) CD34.sup.+, Thy.sup.+                                                                10.sup.4   16/24                                                 (FBM) CD34.sup.+, Thy.sup.-                                                                10.sup.4    2/24                                                 (HDC) CD34.sup.+                                                                           2 × 10.sup.3                                                                       2/5                                                   (FL) HLA-DR.sup.+, Lin*                                                                    10.sup.4   11/14                                                 ______________________________________                                         FBM: Fetal Bone Marrow                                                        Lin* CD3, -8, -10, -15, -19, -20                                              HDC: Human coculture cells grown under Dexterlike conditions for 6 weeks.     FL: Fetal Liver                                                          

The above results show that a small population of selected cells giverise to T-cells, resulting in terminally differentiated CD4⁺ and CD8⁺T-cells. The Thy-1⁻ population does not appear to provide a detectablelevel of differentiated T-cells.

Human bone fragments can be transplanted at various sites (i.p., s.c.,etc.) into either irradiated (200-300 rad) or non-irradiated CB17SCID/SCID mice. The bone fragments grow for periods of at least 9 monthswith the continuous production of human B, myeloid and erythroid cells.The bone fragments act as a supportive microenvironment for humanallogeneic progenitor populations. Allogeneic bone marrow progenitors(mismatch for HLA Class I) may be injected into the bone fragment eitherbefore or after transplantation into the recipient mouse. If theprogenitor cells are to be injected before transplantation, the sortedcells are microinjected into the bone after which the injected bones areincubated overnight at room temperature before transplantation into theCB17 SCID/SCID mice (i.p. or s.c.). If progenitor cells are to bemicroinjected after transplantation, CB17 SCID/SCID mice aretransplanted (i.p., s.c.) with bone fragments. After 6 weeks, the bonegraft can be irradiated (200-1000 rads to the graft with shielding ofthe mouse) followed by injection of progenitor cells. Alternatively, theprogenitor cells may be injected into non-irradiated bone fragments.

Results with the bone marrow assay indicate that CD34+ cells containvirtually all of the bone marrow regenerating capacity. The bone marrowregenerating capacity. Subfractionation of the CD34 population has shownthat the Thy⁺ cells in the CD34 subset (5% of CD34 cells) containvirtually all of the B-cell, myeloid cell and erythroid progenitoractivity in the CD34 fraction. The assay may be successfully performedwith between 10 and 10,000 cells. A summary is shown in Table 9.

                                      TABLE 9                                     __________________________________________________________________________    COMPARISON OF THE DIFFERENTIATION POTENTIAL OF HUMAN PROGENITOR               POPULATIONS                                                                            METHYLCELLULOSE                                                                           CO-CULTURE                                               BONE MARROW                                                                            GM          COLONY T-CELL PROGENITOR                                                                        BONE MARROW                            SUBPOPULATION                                                                          COLONY FREQUENCY                                                                          FREQUENCY                                                                            ASSAY      TRANSPLANT ASSAY                       __________________________________________________________________________    WHOLE BM   1/5,000     1/3000                                                                             ++         ++                                     CD34+    1/500       1/200  ++         ++                                     (2-10%)*                                                                      CD34-      1/50,000    1/50,000                                                                           +/-        +/-                                    (90-98%)                                                                      CD34+Thy+                                                                              1/300       1/20   +++        +++                                    (0.1-0.5%)                                                                    CD34+Thy-                                                                              1/300       1/400  +/-        -                                      (2-10%)                                                                       Thy+Lin+  1/1000     0/854  **         **                                     ((0.3-2.0%)                                                                   Thy+Lin- 1/500       1/21   **         **                                     (0.1-0.5%)                                                                    __________________________________________________________________________     *Percentage represents the percentage of whole bone marrow with the           designated phenotype.                                                         **Experiments underway.                                                  

A secondary transfer assay may be used to evaluate self-renewal andlongevity of human stem cells. Donor progenitor cells are injected intoHLA mismatched bone fragments as above. After 2 to 3 months, theresulting donor cells which have expanded can be resorted for the "stemcell" phenotype (CD34⁺, Thy⁺, Lin⁻). These cells are then reinjectedinto a second HLA mismatched donor. After 1 to 3 months, the bone isevaluated for various progenitor populations as well as stem cells. Inthis way, one can evaluate the long-term potential of "stem cells" togive rise to hematopoietic cells of various lineages as well as thenumber of "stem cells" which arise from a known input "stem cell"number. This provides an estimate of stem cell self-renewal. In thesubject assay, the stem cells of the subject invention would providelong-term renewal.

It is evident from the above results, that the subject inventionprovides for cells which are substantially homogenous in thecharacteristics of human hematopoietic stem cells in accordance withthis invention. Thus, by appropriate selection with particular factorsand the development of bioassays which allow for self regeneration ofstem cells and screening of the stem cells as to their surface markers,a substantially homogenous viable human hematopoietic stem cellcomposition may be produced for a variety of purposes. The stem cellsmay be used in bone marrow transplants, where the cells may be freed ofneoplastic cells or other cells that are pathogenic, e.g., HIV-infectedcells. Further, the use of pure stem cells will precludegraft-versus-host disease. In addition, the cells may be modified byappropriate recombination, either homologous or non-homologous, tocorrect genetic defects or provide genetic capabilities naturallylacking in the stem cells, either as to the individual or as to stemcells generally. In addition, because the composition is substantiallyfree of other cells, the stem cell composition may be used to isolateand define factors associated with regeneration and differentiation.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A culture comprising:human hematopoietic stemcells having fewer than 5% lineage committed cells, wherein said humanstem cells are Thy-1⁺, CD34⁺, capable of self regeneration, and capableof differentiation to members of the lymphoid, erythroid andmyelomonocytic lineages; and a medium capable of supporting the growthof said stem cells.
 2. A culture according to claim 1, wherein saidculture is maintained for at least one week.
 3. A culture according toclaim 1, wherein said culture further comprises stromal cells capable ofsupporting said growth of said stem cells.
 4. A culture according toclaim 3, wherein said stromal cells are mouse cells.
 5. A cultureaccording to claim 1, wherein said medium comprises at least one ofinterleukin 3, interleukin 6 or interleukin
 7. 6. A coculturecomprising:a medium capable of supporting the growth of hematopoieticstem cells; stromal cells; and human hematopoietic cells, including stemcells and differentiated cells, wherein said coculture originated from acellular composition comprising human hematopoletic stem cells havingfewer than 5% lineage committed cells, wherein said human hematopoieticstem cells are Thy-1⁺, CD34⁺, capable of self regeneration, and capableof differentiation to members of the lymphoid, erythroid andmyelomonocytic lineages.
 7. A according to claim 6, wherein said saidcoculture medium comprises hydrocortisone.